Identification of Transcriptional Regulatory Networks in Entamoeba Histolytica

描述（由申请人提供）：转录控制允许参与相似过程或生命阶段的基因协调表达。溶组织内阿米巴是寄生虫死亡的主要原因，但对这种原生动物寄生虫引起侵袭性结肠或肝脏疾病的毒力程序知之甚少。尽管有大量关于模式生物转录调控机制的信息，但在大多数寄生虫系统中对这种机制的了解有限。我们实验室的长期目标是了解寄生虫的遗传和基因表达如何影响毒力。我们的目标是确定控制溶组织芽胞杆菌毒力相关基因亚群表达的转录调控网络。鉴定溶组织芽胞杆菌的转录调控机制将有助于深入了解该寄生虫的基本生物学特性，并为研究阿米巴原虫的基因调控提供途径。在目标1中，我们将确定启动子基序，允许参与毒力的基因协调表达。此外，我们将在感兴趣的基因集的3'UTR和下游基因组区域寻找保守的基序。我们对“毒力”的定义包括半胱氨酸蛋白酶基因（参与组织侵袭）、阿米巴中参与应激反应的基因（Ehssp基因家族）、毒力和非毒力溶组织芽胞杆菌菌株差异表达的基因、结肠炎动物模型体内调控的基因以及寄生虫发育过程中调控的基因。在目标2中，我们将使用报告基因和电泳迁移量转移测定，从功能上表征半胱氨酸蛋白酶、Ehssp和菌株特异性基因家族的潜在调控基元。这些目标的成功将使我们能够证明我们的方法是有效的。在结肠炎动物模型和发育过程中，控制基因表达的基序将在以后的时间里进行表征，因为这些在技术上更具挑战性，并且需要在溶组织大肠杆菌中开发新的技术。这项工作将确定控制与毒力相关的阿米巴基因表达的全球转录调控网络，并为溶组织内阿米巴转录机制的表征提供框架。

DESCRIPTION (provided by applicant): Transcriptional control allows coordinated expression of genes involved in similar processes or life stages. Entamoeba histolytica is a leading cause of parasitic death, but little is known about the virulence program that this protozoan parasite uses to cause invasive colonic or hepatic disease. Although a great deal of information is available on transcriptional regulatory mechanisms in model organisms, there is limited knowledge about such mechanisms in most parasite systems. The long-term goals of our lab are to understand how parasite genetics and gene expression affect virulence. Our goal is to identify transcriptional regulatory networks that control expression of virulence associated gene subsets in E. histolytica. The identification of transcriptional regulatory mechanisms in E. histolytica will give an insight into the basic biology of this parasite, as well as provide avenues for studying the regulation of amebic genes. In Aim 1 we will identify promoter motifs that allow coordinated expression of genes involved in virulence. Additionally, we will look for conserved motifs in the 3'UTR and downstream genomic regions of the gene sets of interest. Our definition of "virulence" includes the cysteine proteinase genes (involved in tissue invasion), genes involved in the stress response in ameba (Ehssp gene family), genes differentially expressed in virulent and non-virulent E. histolytica strains, genes regulated in vivo in an animal model of colitis, and genes regulated during parasite development. In Aim 2, we will functionally characterize potential regulatory motifs from the cysteine proteinase, Ehssp, and the strain-specific gene families using reporter gene and electrophoretic mobility shift assays. Success in these aims will allow us to show proof of concept that our approach is valid. Motifs that control expression of genes regulated during an animal model of colitis and during development will be characterized at a later date as those are technically much more challenging and will require the development of novel technologies in E. histolytica. This work will identify global transcriptional regulatory networks that control expression of amebic genes associated with virulence and provide a framework for characterization of the transcriptional machinery in Entamoeba histolytica. Entamoeba histolytica is an important pathogen and has an impact on human health on a global scale. The main disease manifestations are dysentery and liver abscesses. Although the majority of disease is in developing countries, this parasite can cause infections anywhere that water purification systems get adversely affected. Events such as the Tsunami or upheaval in the political and social infrastructure of a region can allow disease to emerge. We are interested in understanding the molecular mechanisms that the parasite uses to cause disease.